Method of making hypusine peptides

ABSTRACT

The invention relates to novel peptides synthesized according a method utilizing the hypusine reagent:                    
     wherein: Q 1  Q 2  and Q 3  may be the same or different and are amino protective groups, provided that Q 3  is orthogonal to Q 1  and Q 2 ; and 
     Z is a hydroxy protective group, 
     as well as improved methods of peptide synthesis wherein the above-described hypusine reagent is employed to prepare novel hypusine-containing peptides.

RELATED APPLICATION(S)

This application is a continuation of Ser. No. 09/136,472, filed Aug.19, 1998 now ABN which is a continuation-in-part of Ser. No. 08/975,656,filed Nov. 21, 1997, now abandoned, and a continuation-in-part of Ser.No. 09/136,270, filed Aug. 19, 1998 now U.S. Pat. No. 6,248,866 which isa continuation-in-part of Ser. No. 08/962,300, filed Oct. 31, 1997, nowU.S. Pat. No.: 5,973,113, the entire teachings of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Hypusine [N_(ε)-(4-amino-2-hydroxybutyl)lysine], or[2S,9R)-2,11-diamino-9-hydroxy-7-azaundecanoic acid], an unusualnaturally occurring amino acid, having the structure.

was first isolated from bovine brain extracts by Shiba et al. in 1971[Biochim. Biophys. Acta. Vol. 244, pages 523-531 (1971)]. The moleculehas two chiral centers at positions 2 and 9, each of which can beclassified R or S by the Cahn-Ingold-Prelog method. The (2S,9R)diastereomer (B), formed as a post-translational modification

of lysine, has been shown to occur on a precursor protein of theeukaryotic initiation factor 5A (formerly called elF-4D) [Cooper et al.Proc. Natl. Acad. Sci. U.S.A., Vol. 80, pages 1854-1857 (1983); andSafer, Eur. J Biochem., Vol. 186, pages 1-3 (1989)]. This initiationfactor 5A is unique in that it is the only known cellular protein thatcontains the amino acid hypusine (Hpu). In the mid-1970's elF-5A wasshown to stimulate ribosomal subunit joining and to enhance 80 S-boundMet-t-RNA, reactivity with puromycin [Anderson et al., FEBS Lett., Vol.76, pages 1-10 (1977); and Kemper et al., J. Biol. Chem., Vol. 251,pages 5551-5557 (1976)]. Later, in 1983, Cooper et al., supra, suggestedthat a hypusine-modified protein serves as an important initiationfactor in all growing eukaryotic cells. In 1986, Park et al., J. Biol.Chem., Vol. 261, pages 14515-14519 (1986)] isolated the elF-5A proteinfrom human red blood cells and elucidated the amino acid sequencessurrounding the single hypusine residue, asThr-Gly-Hpu-His-Gly-His-Ala-Lys (SEQ ID NO: 1). Furthermore, and mostinteresting because of the potential application to the control of HIVreplication [Bevec et al., J. Proc. Natl. Acad. Sci. U.S.A., Vol. 91,pages 10829-10833 (1994); and Ruhl et al., J. Cell Biol., Vol. 123,pages 1309-1320 (1994)], the synthesis of elF-5A analogues are of greattherapeutic significance.

Since hypusine is specific to elF-5A, antibodies derived fromhypusine-containing peptides could be used to quantitate the levels ofelF-5A directly and with high specificity. Interest in developing anantibody assay of elF-5A to investigate the physiological role of thisimportant initiation factor prompter total synthesis of hypusine and its(2S,9R)-diastereomer [Bergeron et al., J. Org. Chem., Vol. 58, pages6804-6806 (1993)]. The key step in the synthesis involved theN_(ε)-alkylation of N_(ε)-benzyl-N_(α)-carbobenzyloxy-(L)-lysine benzylester with (R)- or (S)- epichlorohydrin to give the respective (2S,9R)-and (2S,9S)-chlorohydrins. Subsequent displacement of the respectivechlorides by cyanide ion provided the protected hypusine skeletons. Thefinal step, hydrogenation over PtO₂ in AcOH, followed by neutralizationand re-acidification, yielded the respective (2S,9S)- and(2S,9R)-hypusine dihydrochlorides. A comparison of the reported hypusineoptical rotation with that of the synthetic (2S, 9R)-hypusine Bconfirmed the stereochemical integrity of both chiral centers throughoutthe synthesis.

Synthetic methodology for accessing hypusine itself exists and it wasdesirable to have a selectively-protected hypusine reagent which couldbe used to incorporate this unusual amino acid into selected peptides.Copending application Ser. No.: 08/962,300, filed Oct. 31, 1997 entitled“Hypusine Reagent for Peptide Synthesis,” now U.S. Pat. No. 5,973,113the entire contents and disclosure of which are incorporated herein byreference, describes a selectively protected hypusine reagent useful forincorporating hypusine into peptides, as well as methods for preparationof the hypusine reagent.

It is an object of the present invention to provide novelhypusine-containing peptides, as well as methods for their synthesisutilizing the above-described hypusine reagent.

SUMMARY OF THE INVENTION

The above and other objects are realized by the present invention, oneembodiment of which relates to novel peptides containing the hypusinemoiety.

More specifically, the present invention relates to novelhypusine-containing peptides synthesized utilizing the hypusine reagent:

wherein: Q₁, Q₂ and Q₃ may be the same or different and are aminoprotective groups, provided that Q₃ is orthogonal to Q₁ and Q₂; and

Z is a hydroxy protective group.

A further embodiment of the invention relates to compounds of structure(2)

 S—Hpu—T  (2)

which may be synthesized using hypusine reagent (1), wherein Hpu is thehypusine amino acid residue, S and T are each independently peptideresidues from zero to about 12 amino acids in length. Compounds of theinvention find utility in the study of biochemical processes involvinghypusine.

Another embodiment of the invention concerns improved methods of peptidesynthesis wherein the above-described hypusine reagent is employed toprepare novel hypusine-containing peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 depict example reaction schemes for preparing peptides ofthe invention. FIGS. 1 and 2 correspond to the chemistry described inExamples 1 through 8.

FIG. 3 is a depiction of a reaction scheme for synthesizing the hypusinereagent of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The method for synthesizing the hypusine reagent comprises:

a. providing an ester of N_(ε)-, N_(α)-diprotected L-lysine, the esterhaving the formula:

 wherein prot and prot′ are N-protective groups which are mutuallyorthogonal and R is the residue of an esterifying alcohol which isorthogonal with respect to prot and prot′,

b. removing prot from N_(ε)of (22) and converting the product to acompound of the formula:

c. converting (24) to a chlorohydrin of the formula:

d. displacing the Cl group of (25) with CN to produce a nitrile of theformula:

e. debenzylating the N_(ε)group and converting the CN group of (26) toan amine group to produce an amino alcohol of the formula:

f. acylating the free amino groups of (27) to provide a di-N-protectedN_(α)-protected L-lysine ester of the formula:

g. removing R and prot′ from (28) to produce a compound of the formula:

 and

h. acylating the free amino group and protecting the OH group to producethe hypusine derivative (1).

The expression “amino protective group” as used herein is intended todesignate groups (Q₁, Q₂ and Q₃) which are inserted in place of ahydrogen atom of an amino group or groups in order to protect the aminogroup(s) during synthesis.

Selection of a suitable amino protecting group will depend upon thereason for protection and the ultimate use of the protected product.When the protecting group is used solely for protection duringsynthesis, then a conventional amino protecting group may be employed.Appropriate amino protecting groups are known in the art and aredescribed, for example, by Bodanszky in Principles of Synthesis,Springer-Verlag, N.Y. (1984); by Ives in U.S. Pat. No. 4,619,915; and inthe various publications on peptide chemistry referred to in the latter.See also Methoden der Organischen Chemie, Houben-Weyl, Vol. 15, No. 1,for protecting groups and Vol. 15, No. 2, for methods of peptidesynthesis. Representative amino protecting groups for synthetic useinclude acyl groups such as tert-butoxycarbonyl, benzyloxycarbonyl,fluorenylmethoxycarbonyl (FMOC), benzoyl, acetyl and the like. Yet otherconventional amino protecting groups for use in synthesis are describedin the literature [Bodanszky, supra, and Ives, supra].

The expression “hydroxyl protective group” as used herein is intended todesignate a group (Z) which is inserted in place of a hydrogen atom ofan OH group or groups in order to protect the OH group(s) duringsynthesis.

The preferred hydroxyl protective groups are the ethers, with the mostpreferred being the tetrahydropyranyl ether.

The term “orthogonal” when used herein to modify the term “protectivegroup(s)” is intended to designate those protective groups in themolecule which are capable of being selectively removed from themolecule in the presence of other protective groups in the moleculewithout affecting the latter.

The various protecting groups for hydroxyl and amino functions discussedabove can be substituted for the, hydroxyl and amino functions in theinstant amino acids/peptides (or their precursor molecules) by methodswell known in the art. Methods for chemical removal of the protectinggroups (when such are not to be retained in the pharmaceutically usefulend product) are likewise well known to the skilled artisan. Typically,amine protecting groups are chemically removed by acidolysis (acidhydrolysis) or hydrogenation, depending on the particular protectinggroup employed. Hydroxyl and carboxyl protecting groups are typicallyremoved chemically by acid or base hydrolysis. Protecting groups whichare incorporated in the pharmaceutical end product must be amenable tohydrolytic or metabolic cleavage in vivo.

Inspection of the structure of hypusine (A) reveals five potentiallyreactive centers: two primary amino and one secondary amino groups, asecondary hydroxyl group and a carboxyl group. In elF-5A, the α-aminonitrogen (N2) required a protecting group which was orthogonal, i.e.,removable under conditions different from those under which the groupsmasking N7 and N12 are removed, to those masking the other twopotentially reactive amines (N7 and N12). Therefore, the N2 nitrogen wasprotected as, e.g., the N-FMOC derivative, while the N7 and N12 amineswere protected as, e.g., the N-CBZ moieties. The 9-hydroxyl was maskedas, e.g., a tetrahydropyranylether. This protection was necessary as thepoorly reactive secondary hydroxyl was expected to cause difficulty withthe anticipated N-acylating agents used in solid phase synthesis[Stewart, The Peptides, Vol. 3, page 170, Gross et al., eds., AcademicPress, New York (1981)].

As shown in FIG. 3, the synthesis preferably begins with thet-butoxycarbonylation of N_(ε)-CBZ-L-lysine t-Bu ester to give (22) in98% yield [Tarbell et al., Proc. Natl. Acad. Sci. USA, Vol. 69, pages730-732 (1972)].

The N_(ε)-CBZ group of (22) was removed by hydrogenation over 10% Pd-Cin ethanol and aqueous HCl to give 23 in 99% yield [Bergmann et al.,Ber. Dtsch., Chem. Abs., Vol. 65, pages 1192-1201 (1932)]. TheN_(ε)-benzyl-N_(α)-BOC-L-lysine t-Bu ester (24) was synthesized from(23) by reductive amination of the liberated N_(ε)amine withbenzaldehyde and sodium cyanoborohydride [Borch et al., J. Am. Chem.Soc., Vol. 93, pages 2897-2904 (1971)].

The earlier synthesis of hypusine [Bergeron et al., supra] developed achiral 4-amino-2-hydroxy butane synthon for accessing the parentmolecule from an L-lysine derivative. In particular, this fragment madeit possible to elaborate the N_(ε)benzyl group of a protected L-lysineinto the N7-N12 structure of hypusine. In the synthesis of the presentinvention, this concept is further exploited. As shown in the reactionscheme depicted in FIG. 3, the subsequent N_(ε)-alkylation of (24) with(S)-epichlorohydrin gave the (2S,9S)-chlorohydrin (25). Displacement ofthe chloride in (25) by cyanide ion afforded the protected (2S,9R)-hypusine skeleton (26). Debenzylation at N7 and conversion of theterminal nitrile in (26) was accomplished by hydrogenation to give theamino alcohol (27) as a diacetate. Acylation of the amino functions ofamino alcohol (27) at N7 and N12 using CBZ groups as protecting groupsprovided di-CBZ-N_(α)-t-BOC-(L)-lysine t-butyl ester (28). Selectiveremoval of the t-butyl ester and N_(α)-BOC protecting groups wasaccomplished with TFA and triethylsilane [Mehta et al., TetrahedronLett., Vol. 33, pages 5441-5444 (1992)] to give the di-CBZ derivative(29). The secondary 9-hydroxyl function was protected astetrahydropyranylether (30) [Bernady et al, J. Org. Chem., Vol. 44,pages 1438-1447 (1979)] and subsequent acylation of the remainingN_(α)-amine function with 9-fluorenylmethyl N-succinimidyl carbonategave the hypusine reagent (31) with the desired protecting groups.Reagent (31) was converted to the dihydrochloride salt of (2S,9R)-hypusine to give identical ¹H NMR and comparable optical rotationwas cited in the art [Bergeron et al., supra] by removing the FMOC groupwith 4-aminomethyl-piperidine [Beyermann et al, J. Org. Chem., Vol. 55,pages 721-728 (1990)] and de-protection of the remaining protectinggroups following a method by Wang et al. [Int. J. Peptide Res., Vol. 40,pages 344-349 (1992)].

In a similar fashion, hypusine reagent molecules of differingstereochemistries may be obtained in a like manner employing startingmaterials of opposite stereochemistries such as (R)-epichlorohydrin.

The novel peptides (2) of the present invention comprise any syntheticpeptide that incorporates within its structure the hypusine moiety,which is synthesized according to a method involving the use of theabove-described hypusine reagent (1).

In Compounds of structure (2), S and T are peptide residues from zero toabout 12 amino acids in length, and preferably, are peptide residuesfrom zero to about six amino acids in length. Most preferably, S and Tare peptides residues from zero to about three amino acids in length Sand T may vary independently in length and in composition of amino acidresidues. The terminal amino acid reside of T may be hydroxylated.Non-limiting examples of peptides of the invention area:

L-Ser-L-Thr-L-Ser-L-Lys-L-Thr-Gly-Hpu-L-His-Gly-L-His-L-Ala-L-Lys (SEQ.ID NO: 2),

L-Cys-L-Thr-Gly-Hpu-L-His-Gly (SEQ IUD NO: 3),

L-Cys-L-Thr-Gly-Hpu-L-His-Gly-OH (SEQ ID NO:6),

Hpu-L-His-Gly,

L-Thr-Gly-Hpu-L-His-Gly (SEQ ID NO: 4),

L-Lys-L-Thr-Gly-Hpu-L-His-Gly (SEQ ID NO: 5),

wherein the Hpu linkage is the (2S,9R)-diastereomer thereof.

Compounds of the invention find utility in the study of biochemicalprocesses involving hypusine, such as in the study of transportmechanisms for elF5A.

The peptides of the invention may be prepared employing conventionalsteps of peptide synthesis except that the above-described hypusinereagent (1) is employed to incorporate the hypusine moiety into thepeptide chain. Conventional peptide synthesis steps are disclosed, forexample, in Moroder et al., “Hormonal Receptors in Digestive TractPhysiology,” G. Rosselin et al., eds., Elsevier/North-Holland BiomedicalPress, Amsterdam, pages 129-135 (1979); and Moroder et al., Z. Physiol.Chem. Vol. 360, pages 787-790 (1979).

The synthesis of peptides is generally carried out through thecondensation of the carboxyl group of an amino acid, and the amino groupof another amino acid, to form a peptide bond. A sequence can beconstructed by repeating the condensation of individual amino acidresidues in stepwise elongation or, in some cases, by condensationbetween two pre-formed peptide fragments (fragment condensation). Insuch condensations, the amino and carboxy groups that are not toparticipate in the reaction must be blocked with protecting groups whichshould be readily introduced, be stable to the condensation reactionsand be removed selectively from the completed peptide. If a peptideinvolves amino acids with side chains that may react duringcondensation, the problem of protection becomes increasingly difficult.A great range of reactive groups and side chains (amino, carboxy, thiol,hydroxy and the like) must be adequately blocked. Their blocking must bestable to unmasking of the α-amino or α-carboxy block for stepwisecondensation and must be readily removed at the final stage, leaving thecompleted peptide moiety intact.

Several methods are known wherein peptides are synthesized in vitro. Theprincipal methodology used for peptide synthesis involves variations ofthe solid-phase methodology developed by Merrifield et al. See, forexample, Erickson et al., “The Proteins,” third edition, Vol. 2, Chapter3, Academic Press, New York (1976). Solid phase peptide synthesisinvolves attachment of a first amino acid to a solid support, such as aresin, followed by sequential addition of subsequent amino acids whichresults in assembly of the peptide chain on the solid support.

Peptides can also be synthesized by related methods involving couplingpeptide fragments to solid supports as discussed by Erickson et al.,supra, pages 268-269. This technique involves the synthesis of smallpeptide segments containing a few amino acids, which segments are thencoupled to each other using fragment condensation techniques to formlarger peptides. Fragment condensation techniques can be combined withstandard solid phase techniques wherein small peptides are attached toresins followed by sequential attachment of single amino acids or otherpeptide segments. Alternatively, sequential attachment of small peptidesto single resin-bound amino acids can also be accomplished. Thecombination of the two approaches provides flexibility to syntheticschemes.

Upon completion of a particular synthesis, the synthesized peptide isthen removed from the resin, usually by chemical means such as treatmentwith hydrofluoric acid (HF). The chemical treatment also removes variousamino acid and peptide protecting groups, such as CBZ, t-BOC or tosyl,which mask the reactivity of amino acid functional groups duringsynthesis.

In most peptide syntheses, the initial attachment to the resin involvesthe C-terminal amino acid of the peptide to be synthesized, which aminoacid is covalently attached to the resin through an ester or amidelinkage involving its α-carboxyl group. Synthesis then proceeds from theC- to the N-terminal. N-terminal to C-terminal peptide synthesis is lessfrequently used because the chemistry is more difficult and unwantedside reactions are more common.

The first amino acid may be covalently attached to the resin, in somecases, through its functional side chain. Initial attachment of an aminoacid to the resin by means of the side chain functional group allows thepossibility of bi-directional synthesis starting with the attached aminoacid. Bi-directional synthesis cannot be performed if the initial aminoacid is attached through the α-COOH or (α-NH₂ group. Side chainfunctional groups which have been used for attachment to resins includethe sulfhlydryl group of cysteine, the imidazole group of histidine, theδ-amino group of ornithine, the ε-amino group of lysine and theγ-carboxyl group of glutamic acid. A review of the chemistry of solidphase peptide synthesis, including attachment of amino acids to resinsvia the α-COOH, α-NH and functional side chain groups, is found inErickson et al., supra.

By using the insoluble resin support, it is possible to isolate theproduct of each coupling reaction simply by filtering the resin andwashing it free of by-products and excess starting materials. In fact,the synthetic processes are so simplified and the time required for onecycle is so shortened that in recent years, it has become quite commonto use automated peptide synthesizers. [See, for example, Barany et al.,“The Peptides,” Vol. 2, Academic Press, Inc., New York (1979), pages1-284; or Kemp-Vellaccio, “Organic Chemistry,” pages 1030-1032 (1980).]

Although the hypusine reagent described herein may be employed to accessany hypusine-containing peptide, the method of the invention will beillustrated with reference to the following syntheses. It will beunderstood that any conventional peptide synthesis may be modified toprepare a hypusine-containing peptide by simply utilizing the hereindescribed hypusine reagent at any convenient stage thereof.

While the hypusine reagent allows for the assembly of a variety ofhypusine-containing peptides, the hypusine-containing pentapeptide foundin elF-5A capped at its N-terminus with L-Cys, i.e.,L-Cys-Thr-Gly-Hpu-His-Gly (SEQ ID NO: 3) is a typical target peptide.The L-Cys, which is not contained in the natural peptide, was fixed tothe sequence with the idea of being able to covalently link the peptidevia a disulfide bond to a larger protein, in order to ultimatelygenerate antibodies. The solution synthesis, a convergent approach, wascarried out by elaborating from the C- to the N-terminus, as shown inFIG. 1, and involved the coupling of three appropriately-protectedpieces: Cys-Thr-Gly, Hpu and His-Gly.

The carboxyamidomethyl (CAM) ester developed by Martinez et al.,[Tetrahedron, Vol. 41, pages 739-743 (1985); and Tetrahedron Lett., Vol.47, pages 5219-5222 (1983)] was employed as a carboxyl protecting groupin generating the Cys-Thr-Gly fragment. This protecting group isorthogonal to the BOC, CBZ and FMOC groups. Thus, the synthesis of themasked Cys-Thr-Gly fragment 15 began with the N-BOC-Gly-CAM ester[Martinez et al., Tetrahedron, supra]. Removal of the BOC group withtrifluoroacetic acid (TFA) gave the amine salt (60%) which wasimmediately coupled with N-FMOC-(O-t-butyl)-L-threonine to give theN-FMOC-(O-t-butyl)-L-Thr-Gly-CAM ester 13 in 85% yield. Treatment of 13with 10% diethylamine (DEA) in DMF followed by coupling with N,S-di-CBZ-L-cysteine with BOP and DIEA afforded the N,S-di-CBZ-L-Cys-(O-t-butyl)-L-Thr-Gly-CAM ester 14 in 50% yield. Removalof the CAM ester with aqueous Na₂CO₃ followed by acidification withaqueous citric acid generated the N,S-di-CBZ-L-Cys-(O-t-butyl)-L-Thr-Gly acid 15 in 77% yield.

The design of the His-Gly fragment 17 was predicated on obtainingefficient coupling between the N₆₀ His group and reagent 1. Previouswork by Yamashiro et al., [J. Am. Chem. Soc., Vol. 94, pages 2855-2859(1972)] demonstrated that t-butyl-carbonylation of the imidazole sidechain prior to the condensation step substantially increased couplingyields between systems containing His residues and N-BOC groups. Forthis reason, the His-Gly fragment 17 was assembled in two steps. First,the condensation of glycine t-butyl ester and N_(α)-CBZ-L-His withdiphenyl-phosphorylazide (DPPA) [Shioiri et al., J. Am. Chem. Soc., Vol.94, No. 17, pages 6203-6205 (1972)] afforded N_(α)-CBZ-L-His-Gly t-butylester 16 (72%). In the second step, the His side chain was masked by thetreatment with di-t-butyl dicarbonate in TUF to give theN_(α)-CBZ-N_(im)-BOC-L-His-Gly t-butyl ester 17 in 78% yield.

As shown in FIG. 1, the final hexapeptide 12 was constructed stepwisefrom the three aforementioned fragments, i.e., 1, 15 and 18.Hydrogenolysis of the N_(α)-CBZ group of 17 provided the amine HCl salt(74%) which was condensed with hypusine reagent 1 to give the di-CBZ-THPprotected Hpu-His-Gly tripeptide 18 in 85% yield. The amine generated bytreatment of the N_(α)-FMOC in 18 with 4-aminomethyl-piperidine (68%)[Beyermann et al, J. Org. Chem., Vol. 55, pages 721-728 (1990)] wasacylated by the tripeptide acid 15 and BOP to give the masked conjugate19 in 81% yield. The final deprotection of 19 with HBr/acetic acid inTFA and a “cocktail” of scavengers (phenol, pentamethylbenzene,triisopropylsilane, 1,2-ethanedithiol) at room temperature developed byWang et al., [Int. J. Peptide. Res., Vol. 40, pages 344-349 (1992)] gavethe Cys-Thr-Gly-Hpu-His-Gly (SEQ ID NO: 3) as itstetrakis-trifluoroacetic acid salt 12 in 22% yield. The peptide wasfully assigned by DOCOSY, TOCSY and HMOC NMR.

Polymer bounded synthesis of the hexapeptide 12 was performed on aHMP-resin [Want, supra] using an Applied Biosystems 432A Synthesizer.The Cys and His residues of the hexapeptide were initially protectedwith trityl groups while the Thr was protected as its t-butyl ether.Attempts to release the free hexapeptide by refluxing in TFA/phenol orwith HBr/acetic acid in TFA with phenol and pentamethyl-benzene,respectively, did not succeed. Use of the more labile 4-methoxytrityland 4-methyltrityl groups to protect the Cys and His derivatives,respectively, produced the protected hexapeptide 20 (FIG. 2). Finaldeprotection of 20 was then achieved with HBr/acetic acid in TFA using a“cocktail” of four cation scavengers. This method generated very fewside products and provided 12 after purification by HPLC in 24% yield.All analytical data were consistent with hexapeptide 12*4 TFA previouslyprepared by the solution-phase method.

In summary, the hypusine reagent described has been demonstrated to be ahighly useful synthon in accessing the elF-5A pentapeptide sequence.While the yields are generally excellent for these kinds of systems, themost notable feature is the flexibility that this methodology offers insynthesizing related elF-5A mimics.

EXAMPLE 1 FMOC-Thr(O-t-butyl)-Gly Carboxyamido Methyl (CAM) Ester (14)

N_(α)-BOC-Gly CAM ester [Martinez et al., Tetrahedron, supra] (1.16 g,5.0 mmol) was dissolved in TFA (10 ml) and stirred 10 minutes at 0° C.The amine TFA salt was precipitated with diethyl ether (130 ml),filtered and dried (0.75 g, 60%). The amine salt (0.75 g) was thendissolved in a dry DMF (10 ml) solution containingFMOC-Thr(O-t-butyl)-OH (1.20 g, 3.0 mmol) and BOP (1.33 g, 3.0 mmol).The solution was cooled to 0° C., and diisopropylethylamine (DIEA, 1.15ml, 6.6 mmol) was added dropwise. The solution was warmed to roomtemperature and stirred overnight. The volatiles were removed in vacuo,the resulting oil was dissolved in ethyl acetate (200 ml) and washedwith 1 M citric acid, water, 5% aqueous NaHCO₃ solution and water. Theorganic layer was dried over MgSO₄ and concentrated. Flashchromatography (90% ethyl acetate/hexane) gave 14 as a colorless solid(1.30 g, 85%). ¹H NMR (CDCl₃) δ 7.77 (d, 2H, J=7.5 Hz), 7.65 (m, 1H),7.60 (d, 2H, J=7.5 Hz), 7.45-7.27 (m, 4H), 6.60 (s br, 1H), 5.87 (d br,1H), 5.54 (s br, 1H), 4.67 (m, 2H), 4.51-4.35 (m, 2H), 4.28-4.12 (m,4H), 4.05 (m, 1H), 1.29 (s, 9H), 1.08 (d, 3H, J=6.4 Hz). Anal. calcd.for C₂₇H₃₃N₃O₇: C63.39; H, 6.50, N, 8.21. Found: C63.14; H, 6.60, N,8.10. [α]²² _(D)−0.8° (c=1.00, CH₃OH).

EXAMPLE 2 CBZ-Cys(CBZ)-Thr(O-t-butyl)-Gly Carboxyamidomethyl Ester (15)

A solution of 14 (0.46 g, 0.90 mmol) was dissolved in a solution ofdiethylamine (DEA, 0.8 ml) in dry DMF (8 ml). After stirring overnightat room temperature, the volatiles were removed in vacuo and the residuedissolved in 15 ml dry DMF. N,S-di-CBZ-L-Cys (0.35 g, 0.90 mmol) and BOP(0.40 g, 0.90 mmol) were added and the turbid solution cooled to 0° C.DIEA (0.25 g, 1.89 mmol) was added, and the solution was stirred at roomtemperature for 2 days. The reaction mixture was concentrated in vacuo,diluted with ethyl acetate (100 ml) and washed with 10% citric acid,water, 5% aqueous NaHCO₃ solution and water. The organic layer was driedover MgSO₄ and concentrated in vacuo. The resulting oil was purified byflash column chromatography (80% ethyl acetate/10% hexane/10% CHCl₃ toobtain 15 (0.29 g, 50%). mp 95-97° C. ¹H NMR (CDCl₃) δ 7.70 (m, 1H),7.28 (s, 10H), 6.78 (s, 1H), 6.00 (s, 1H), 5.50 (s, 1H), 5.23 (s, 2H),5.17 (s, 2H), 4.62 (m, 2H), 4.41 (m, 1H), 4.30 (m, 1H), 4.25 (m, 1H),4.15 (m, 1H), 3.94 (m, 1H), 3.30 (m, 2H), 1.65 (s, 1H), 1.20 (s, 9H),1.07 (d, 3H), Anal. calcd. for C₃₁H₄₀N₄O₁₀S: C56.35; H, 6.10; N, 8.48.Found: C56.62; H, 6.14, N, 8.38. [α]²⁵ _(D)−14° (c=1.10, CH₃OH).

EXAMPLE 3 CBZ-Cys(CBZ)-Thr(O-t-butyl)-Gly-OH (16)

To a solution of 15 (0.90 g, 1.36 mmol) dissolved in DMF (14 ml) wasadded aqueous Na₂CO₃ (2.72 mmol, 8 ml) at room temperature. The solutionwarmed upon addition and water was added until the solution was clear.After 5 minutes, the pH was adjusted to 6 with citric acid (0.5 N, 9ml). The reaction mixture was concentrated in vacuo; the residue wasdissolved in ethyl acetate and washed with 0.5 N citric acid. Theorganic layer was dried over sodium sulfate and concentrated in vacuo.The residue was purified by column chromatography using LH-20 lipophilicSephadex (50 g; eluting with 1% EtOH/toluene) to give 16 as a whitesolid (0.63 g, 77%). mp 124-125OC; ¹H NMR (CD₃OD) δ 7.39-7.25 (m, 10H),5.24 (s, 2H), 5.12 (s, 2H), 4.46 (dd, 1H, J=8.6, 5.0 Hz), 4.33 (d, 1H,J=3.0 Hz), 4.16 (m, 1H), 3.92 (m, 2H), 3.45 (dd, 1H, J=14.3, 5.0 Hz),3.16 (dd, 1H, J=14.3, 8.6 Hz), 1.20 (s, 9H), 1.09 (d, 3H, J=6.4 Hz).Anal. Calcd. for C₂₉H₃₇N₃O₉S: C57.70; H, 6.18; N, 6.96. Found: C57.76;H, 6.24; N, 7.02. [α]²² _(D)+8.6° (c=0.50, CHCl₃).

EXAMPLE 4 CBZ-His-Gly-O-tert-Butyl (17)

Glycine t-butyl ester hydrochloride (2.20 g, 13.0 mmol) andN_(α)-CBZ-L-His (3.44 g, 11.9 mmol) were dissolved in dry DMF (40 ml).The solution was cooled to 0° C. under an N₂ atmosphere, anddiphenylphosphoryl azide (DPPA, 3.58 g, 13.0 mmol) was added dropwiseover 5 minutes. The clear solution was stirred for 10 minutes and turnedturbid upon addition of triethylamine (2.63 g, 26 mmol). The solutionwas stirred at room temperature overnight. The solution was concentratedand the residue purified by flash chromatography (8% EtOH/CHCl₃) to give17 as a white solid (3.45 g, 72%). mp 64-66° C. ¹H NMR (CD₃OD) δ 7.56(d, 1H, J=1.1 Hz), 7.30 (m, 5H), 6.88 (s, 1H), 5.04 (m, 2H), 4.42 (m,1H), 3.80 (m, 2H), 3.12 (m, 1H), 2.91 (m, 1H), 1.46 (s, 9H). Anal.calcd. for C₂₀H₂₆N₄O₅: C59.69; H, 6.51; N, 13.92. Found: C59.43; H,6.46; N, 13.81.

EXAMPLE 5 CBZ-His(BOC)-Gly-O-tert-Butyl (18)

A solution of di-t-butyl dicarbonate (1.33 g, 6.0 mmol) in 3 ml THF wasadded dropwise at 0° C. to a solution of 17 (2.0 g, 5.0 mmol) in 15 mlTHF. The reaction mixture was stirred overnight at room temperature andconcentrated in vacuo. The residue was purified by flash chromatography(60% ethyl acetate/hexane) to give 1.96 g of 18 (78%). ¹H NMR (CDCl₃) δ8.00 (s, 1H), 7.30 (m, 6H), 6.60 (m, 1H), 5.10 (s, 2H), 4.58 (m, 1H),3.85 (m, 2H), 3.03 (m, 2H), 2.38 (s, 1H), 1.60 (s, 9H), 1.40 (s, 9H).Anal. Calcd. for C₂₀H₂₆N₄O₅: C59.75; H, 6.82; N, 11.15. Found: C59.69;H, 6.87; N, 11.09. [α]²² _(D)−4.0° (c=1.00, CH₃OH).

EXAMPLE 6 FMOC-Hpu(N7, N12-di-CBZ,09-THP)-His(BOC)-Gly-O-tert-Butyl (19)

To a solution of 18 (185 mg, 0.37 mmol) and 1 N CHl (370 μl) in ethanol(15 ml) was added 10% Pd-C (18 mg), and the reaction mixture washydrogenated for 2.5 hours at room temperature. The reaction mixture wasfiltered through Celite, concentrated in vacuo and purified by flashchromatography (CHCl₃/EtOH=9:1) to give H-His-(BOC)-Gly-O-t-Buhydrochloride (101 mg, 68%). A portion of this ammonium salt (69 mg,0.17 mmol) and hypusine reagent 1 (134 mg, 0.17 mmol) were dissolved in12 ml dry DMF. The solution was cooled to 0° C. and BOP (87 mg, 0.20mmol) was added and stirred for 20 minutes. DIEA (47.5 mg, 0.37 mmol)was added dropwise at 0° C. and the solution warmed to room temperatureand stirred overnight. The volatiles were removed under reducedpressure, and the residue was dissolved in ethyl acetate and washed with5% aqueous NaHCO₃ solution and water. The organic layer was dried overMgSO₄ and concentrated in vacuo and the residue purified by flashchromatography (4% EtOH/CHCl₃) to give 19 as a colorless oil (165 mg,85%). ¹H NMR (600 MHz) (CD₃OD) δ 7.86 (m, 1H), 7.70 (d, 2H, J=7.5 Hz),7.58 (m, 2H), 7.31-7.11 (m, 15H), 5.00 (m, 2H), 4.92 (s, 2H), 4.60 (m,1H), 4.50-4.32 (m, 1H), 4.30-4.26 (m, 2H), 4.08 (t, 1H, J=6.8 Hz), 3.90(m, 1H), 3.86-3.52 (m, 5H), 3.38-2.76 (m, 8H), 1.70-1.12 (m, 14H), 1.40(s, 9H), 1.28 (s, 9H). HRMS m/z calcd. for C₆₃H₈₀N₇O₁₄ 1158.5763; found1158.5739. [α]² _(D)−1.5° (C=1.00, CHCl₃).

EXAMPLE 7CBZ-Cys(CBZ)-Thr(O-t-Bu)-Gly-Hpu(N7,N12-di-CBZ,O9-THP)-His(BOC)-Gly-O-t-Bu(20)

4-(Aminomethyl)-piperidine (1.0 ml) was added to 19 (156 mg, 0.14 mmol)dissolved in CHCl₃ (10 ml). The clear solution was stirred for 2 hoursat room temperature. An additional portion of 4-(aminomethyl)-piperidine(1.0 ml) was added and stirring was continued another 40 minutes. Thereaction mixture was taken up in 50 ml CDCl₃ and extracted three timeswith phosphate buffer (pH=5.5, 75 ml each). The organic layer was driedover Na₂SO₄, concentrated and purified by flash chromatography on silica(10% MeOH/CHCl₃) to give H-Hpu(N7,N12-di-CBZ,O9-TBP)-His(BOC)-Gly-O-t-Bu as a colorless oil (88 mg,68%). A portion of the colorless oil (10 mg, 11 μmol) and 16 (7 mg, 11μmol) in dry DMF (2 ml) was cooled to 0° C. BOP (6 mg, 12 μmol) wasadded and stirred for 30 minutes. Diisopropylethylamine (4.5 μl, 26μmol) was added dropwise and the solution was warmed to room temperatureand stirred overnight. The reaction mixture was concentrated in vacuo.The residue was dissolved in ethyl acetate (15 ml) and extracted with 5%aqueous NaHCO₃ solution (10 ml) and water (10 ml). The organic layer wasconcentrated under reduced pressure and the residune purified by flashchromatography (10% EtOH/CHCl₃; Rf=0.35) to give 20 as a colorless oil(13 mg, 81%). ¹H NMR (600 MHz) (CD₃OD) δ 7.99 (m, 1H), 7.28-7.14 (m,21H), 5.17-5.07 (m, 2H), 5.06-4.88 (m, 6H), 4.58 (dd, 1H, J=9.0, 4.2Hz), 4.51-4.32 (m, 2H), 4.26 (m, 1H), 4.14 (m, 1H), 4.10 (m, 1H),4.08-3.98 (m, 2H), 3.86 -3.58 (m, 6H), 3.40-2.94 (m, 8H), 2.88 (m, 1H),1.70-0.94 (m, 17H), 1.48 (s, 9H), 1.34 (s, 9H), 1.08 (s, 9H). HRMS m/zcalcd. for C₇₇H₁₀₅N₁₀O₂₀S 1521.7227; found 1521.736. [α]²⁶ _(D)−1.2°(c=1.00, CHCl₃).

EXAMPLE 8 Cys-Thr-Gly-Hpu-His-Gly (SEQ ID NO: 3), Trifluoroacetic AcidSalt (12)

Method (a)

A solution of 20 (13 mg, 7.2 μmol) and phenol (5 mg, 50 μmol) was heatedto reflux for 90 minutes in degassed TFA (5.0 ml) under an argonatmosphere. The reaction mixture was concentrated in vacuo and theresidue applied to a C-18 plug (Supelco; water/acetonitrile=88/22+0.1%TFA). Further purification was performed by preparative HPLC (solventsystems A, aqueous 0.1% TFA; and B, 0.1% FA in CH₃CN; linear gradient of0-20% B in 50 minutes; flow rate 4.0 ml/minute; detection at 214 nm;retention time=8.4 minutes) using a C-18 reverse phase column (Dynamax300 A C₁₆) to give 12 as a colorless oil (2 mg, 24%). ¹H NMR (D₂O) (600MHz) δ 8.69 (d, 1H, J=1.2 Hz), 7.38 (s, 1H), 4.80 (m, 1H), 4.49 (d, 1H,J=4.9 Hz), 4.41 (t, 1H, J=5.6 Hz), 4.36 (dd, 1H, J=8.5, 6.0 Hz), 4.29(m, 1H), 4.11 (m, 1H), 4.09-4.00 (m, 4H), 3.37 (dd, 1H, J=15.5, 7.2 Hz),3.29-3.06 (m, 9H), 1.99 (m, 1H), 1.86 (m, 2H), 1.76 (m, 3H), 1.45 (m,2H), 1.32 (d, 3H, J=6.4 Hz). MS (MALDI-Tof) m/z calcd. for C₂₇H₄₈N₁₀O₉S688.33 (M⁺), found 688.96.

Method (b)

The polymer-bound peptide 21 was synthesized using an Applied Biosystems432A Synthesizer. Amino acid analysis for 21: Gly 2.09, His 1.03, Thr0.88. An aliquot of 21 (49 mg, 19.3 , μmol), phenol (250 mg) andpentamethylbenzene (250 mg) were dissolved in degassed TFA (5.0 ml) at0° C. Saturated HBr in acetic acid solution (0.2 ml), triisopropylsilane(0.1 ml) and 1,2-ethanedithiol (0.1 ml) were added under an argonatmosphere. The solution was stirred at room temperature for 1 hour andconcentrated under reduced pressure. The residue was dissolved in 10%acetic acid (10 ml) and extracted with methyl tert-butyl ether (3×25ml). The aqueous layer was concentrated in vacuo and the residue waspurified on a preparative HPLC as in method (a) above using a C-18reverse phase column (Dynamax 300 A C₁₈) to give 12 as a colorless oil(5.2 mg, 24%). ¹H NMR and MS analytical data were identical to those for12 prepared by method (a) above. [α]²⁶ _(D)−167° (c=0.30, H₂O). Aminoacid analysis: Gly 1.94, His 1.02, Thr 1.04.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

EXAMPLE 9 N_(α)-BOC-N_(ε)-CBZ-L-Lysine tert-Butyl Ester (22)

Sodium hydrogencarbonate (2.81 g, 33.47 mmol) in water (75 ml) was addedto H-Lys(CBZ)-O-t-Bu hydrochloride (12.00 g, 32.18 mmol) in chloroform(100 ml) and the mixture was stirred at room temperature for 5 minutesunder an N₂ atmosphere. Di-tert-butyl dicarbonate (7.02 g, 32.18 mmol)in chloroform (50 ml) was added, the mixture refluxed for 1.5 hours andallowed to cool to room temperature. The layers were separated, theaqueous layer extracted with chloroform (3×100 ml) and the combinedorganic layers dried over magnesium sulfate. Concentration in vacuofollowed by flash chromatography (3:1 hexane:ethyl acetate) gave (22)(13.82 g, 98%) as a colorless oil. ¹H NMR (CDCl₃) δ 7.30 (s, 5H), 5.10(s, 2H), 4.82 (m, 1H), 4.18 (m, 1H), 3.20 (m, 2H), 1.90-1.30 (m, 6H),1.48 (s, 9H), 1.46 (s, 9H); ¹³C NMR (CD₃OD) δ 173.8, 158.8, 158.1,138.4, 129.4, 128.9, 128.7, 82.5, 80.4, 67.3, 55.7, 41.4, 32.4, 30.4,28.7, 28.3, 24.0. HRMS m/z calcd. for C₂₃H₃₇N₂O₆ 437.2652, found437.2643. Anal. calcd. for C₂₃H₃₆N₂O₆: C63.28, H, 8.31, N, 6.42. Found:C63.13, H, 8.28, N, 6.47. [α]²⁷ _(D)+5.0 (c=2.00, CHCl₃).

EXAMPLE 10 N_(α)-BOC-L-Lysine tert-Butyl Ester Hydrochloride (23)

N_(α)-BOC-N_(ε)-CBZ-L-lysine tert-butyl ester (22) (34.51 g, 79.15 mmol)was dissolved in a mixture of 300 ml absolute EtOH and 1 N HCl (88 ml).Prior to the introduction of H₂ gas, 10% Pd-C (2.95 g) was added. After7 hours, additional catalyst (1.0 g) was added. After 5 hours, the blacksuspension was filtered through a bed of Celite and washed with EtOH.The filtrate was concentrated and the residue dried under high vacuum togive the N_(α)-BOC-L-lysine tert-butyl ester as its hydrochloride salt(23) (26.59 g, 99%). ¹H NMR (CD₃OD) δ 3.95 (dd, 1H, J=8.8, 5.0 Hz), 2.93(t, 2H, J=7.7 Hz), 1.84-1.60 (m, 6H), 1.45 (s, 9H), 1.43 (s, 9H); ¹³CNMR (CD₃OD) δ 173.5, 15 8.2, 82.7, 80.5, 79.5, 5 5.5, 40.6, 32.1, 28.7,28.3, 23.9. HRMS m/z calcd. for C₁₅H₃₁N₂O₄ 303.2284, found 303.2272.[α]²⁶ _(D)−10.1° (c=1.00, CH₃OH).

EXAMPLE 11 N_(ε)-Benzyl-N_(α)-BOC-L-Lysine tert-Butyl Ester (24)

N_(α)-BOC-L-lysine t-butyl ester hydrochloride salt (23) (25.97 g, 76.64mmol) was dissolved in CHCl₃ (300 ml) and washed with saturated aqueousNa₂CO₃ solution (2×100 ml). The organic layer was separated, dried(MgSO₄), filtered and concentrated. The resultant oil (the free amine)was combined with benzaldehyde (10.42 g, 98.13 mmol), EtOH (150 ml) andactivated 3 Å molecular sieves (46.0 g). The mixture was stirred underN₂ for 6 hours. Sodium cyanoborohydride (2.41 g, 3 8.4 mmol) was addedand the mixture was stirred overnight at room temperature. The brownmixture was filtered and the filtrate acidified to pH, 2 with 1 N HCl(110 ml). The yellow solution was concentrated to dryness, dissolved inCHCl₃, washed with saturated Na₂CO₃ solution and water. The organiclayer was separated, dried (MgSO₄) and concentrated. Flash columnchromatography (10% EtOH/CHCl₃, R_(f)=0.30) afforded theN_(ε)-benzyl-N_(α)-BOC-L-lysine t-butyl ester (24) (16.16 g, 54%) as acolorless oil. ¹H NMR (CD₃OD) δ 7.34-7.20 (m, 5H), 3.91 (dd, 1H, J=9.0,5.1 Hz), 3.72 (s, 2H), 2.58 (t, 2H, J=7.2 Hz), 1.82-1.30 (m, 6H), 1.45(s, 9H), 1.43 (s, 9H); ¹³C NMR (CDCl₃) δ 171.9, 155.3, 140.1, 128.3,128.1, 126.9, 81.6, 79.5, 53.9, 48.9, 32.7, 29.5, 28.3, 27.9, 22.9. HRMSm/z calcd. for C₂₂H₃₆N₂O₄ 392.2675, found 392.2676. Anal. calcd. forC₂₂H₃₅N₂O₄: C67.32, H, 9.24, N, 7.14. Found: C67.40, H, 9.28, N, 7.16.[α]²⁵ _(D)+6.9° (c=1.00, CDCl₃).

EXAMPLE 12(2S,9S)-7-Benzyl-2-[(tert-butoxycarbonyl)amino]-10-chloro-9-hydroxy-7-azadecanoicAcid, tert-Butyl Ester (25)

A mixture of N_(ε)-benzyl-N_(α)-BOC-L-lysine t-butyl ester (24) (16.0 g,40.76 mmol), CH₃OH (40 ml), (S)-(+)-epichlorohydrin (4.17 g, 45.0 mmol)and anhydrous MgSO₄ (5.33 g. 44.28 mmol) was stirred under N₂ for threedays. The solids were filtered off and washed with CH₃OH. The filtratewas concentrated at room temperature to give a yellow oil. The resultingoil was purified by flash chromatography on silica gel (66% hexane/ethylacetate) to give 13.23 g of (25) (77%) as a colorless oil. ¹H NMR (C₆D₆)δ 7.18 (m, 5H), 5.00 (br d, 1H), 4.40 (m, 1H), 3.62 (m, 1H), 3.40-3.10(m, 4H), 2.20-2.00 (m, 4H), 1.63 (m, 1H), 1.40 (s, 9H), 1.20 (m, 2H).¹³C NMR (C₆D₆) δ 171.7, 155.2, 138.8, 128.8, 127.9, 127.0, 80.8, 78.8,67.7, 58.8, 57.2, 53.9, 53.7, 47.3, 32.5, 28.0, 27.5, 26.3, 22.8. HRMSm/z calcd. for C₂₅H₄₂ClN₂O₅ 485.2782, found 485.2775. [α]²⁵ _(D)+5.3°(c=1.00, CHCl₃).

EXAMPLE 13(2S,9R)-7-Benzyl-2-[(tert-butoxycarbonyl)amino]-10-cyano-9-hydroxy-7-azadecanoicAcid tert-Butyl Ester (26)

A mixture of (25) (6.99 g, 14.4 mmol), dry KCN (9.38 g, 144 mmol) and18-crown-6 (0.76 g, 2.88 mmol) in 275 ml of dry acetonitrile was stirredat 45° C. for 5 days. It should be noted that heating this mixture toreflux causes significant decomposition. The reaction mixture wascooled, filtered and concentrated. Flash column chromatography on silicagel (25% ethyl acetate/hexane) gave the (2S,9R)-nitrile (26) as acolorless oil (4.82 g, 70%). ¹H NMR (CD₃OD) δ 7.34-7.18 (m, 5H),3.97-3.83 (m, 2H), 3.67 (dd, 1H, J=13.4, 2.6 Hz), 3.54 (dd, 1H, J=13.4,4.0 Hz), 2.72-2.40 (m, 6H), 1.80-1.50 (m, 4H), 1.45 (s, 9H), 1.44 (s,9H), 1.40-1.30 (m, 2H). ¹³C NMR (CDCl₃) δ 171.8, 155.3, 138.1, 128.8,128.4, 127.3, 117.1, 81.6, 79.5, 63.6, 58.8, 54.0, 32.6, 28.2, 27.9,22.1. HRMS m/z calcd. for C₂₆H₄₂N₃O₅ 476.3124, found 476.3121. Anal.calcd. for C₂₆H₄₁N₃O₅: C65.66, H, 8.69, N, 8.83. Found: C65.71, H, 8.67,N, 8.80. [α]²⁵ _(D)+4.7° (c=1.00, CHCl₃).

EXAMPLE 14 (2S,9R)-2-[(tert-Butoxycarbonyl)amino]-l-amino-9-hydroxy-7-azaundecanoic Acid tert-Butyl Ester, Diacetate Salt(27)

The N_(ε)benzyl nitrile (26) (4.80 g, 10.7 mmol) was dissolved inglacial acetic acid (100 ml); 10% Pd-C (0.50 g) and PtO₂ (1.00 g) wereadded; and hydrogen gas was introduced. The reaction was complete after6 hours, and the catalyst was filtered through a bed of Celite andwashed with acetic acid. The filtrate was concentrated in vacuo.Azeotropic removal of the acetic acid with toluene gave (27) as acolorless oil (5.10 g, 99%) 1H NMR (500 MHz) (CD₃OD) d 4.02-3.94 (m,2H), 3.14-2.86 (m, 6H), 1.94 (s, 6H), 1.87-1.58 (m, 8H), 1.46 (s, 9H),1.44 (s, 9H), ¹³C NMR (CD₃OD) δ 169.6, 156.54, 85.3, 70.2, 62.6, 56.6,54.2, 53.9, 34.0, 31.1, 28.2, 26.3, 23.2. HRMS m/z calcd. for C₁₉H₄₀N₃_(O) ₅ 390.2968, found 390.2977. [α]² _(D+)0.6° (c=1.00, CH₃OH).

EXAMPLE 15(2S,9R)-11-[(Benzyloxycarbonyl)amino]-2-[(tert-butoxycarbonyl)amino]-9-hydroxy-7-carbobenzyloxy-7-azaundecanoicAcid tert-Butyl Ester (28)

A solution of (27) (1.17 g, 2.30 mmol) in CHCl₃ (100 ml) was washed withsaturated Na₂CO₃ solution. The aqueous layer was extracted with CHCl₃(3×100 ml) and the combined organic layers were dried over Na₂SO₄ andconcentrated in vacuo. A solution of the resultant oil (the free amine,0.85 g, 2.18 mmol) in CH₂Cl₂ (60 ml) was cooled to 0° C. and treatedwith diisopropylethylamine (0.59 g, 4.57 mmol) and benzyl chloroformate(0.79 g, 4.60 mmol). The reaction mixture was stirred overnight at roomtemperature, concentrated to dryness and purified by flashchromatography (50% ethyl acetate/hexane) to give (28) (790 mg, 55%) asa colorless oil. ¹H NMR (CDCl₃) δ 7.23 (m, 10H), 5.45 (m, 1H), 5.08 (s,211), 5.04 (s, 2H), 4.10 (m, 1H), 3.80 (m, 1H), 3.40 (m, 1H), 3.23 (m,5H), 1.80-1.43 (m, 6H), 1.41 (s, 18H), NMR (CDCl₃) δ 171.8, 157.5,156.9, 155.3, 136.4, 128.4, 128.3, 127.9, 127.7, 81.6, 79.5, 69.2, 67.2,66.5, 53.7, 48.5, 37.7, 34.8, 32.5, 28.2, 27.9, 22.3. HRMS m/z calcd.for C₃₅H₅₂N₃O₉ 658.3703, found 658.3774. [α]²⁴ _(D)+4.6° (c=0.50,CHCl₃).

EXAMPLE 16(2S,9R)-11-[(Benzyloxycarbonyl)amino]-7-(carbobenzyloxy)-9-hydroxy-7-azaundecanoicAcid (29)

The ester (28) (500 mg, 0.76 mmol) was dissolved in a pre-made mixtureof trifluoroacetic acid (1.12 g, 9.90 mmol), CH₂Cl₂ (2.05 g, 24.0 mmol)and triethylsilane (220 mg, 1.9 mmol) and stirred at room temperaturefor 20 hours. The reaction mixture was concentrated to dryness andstirred again in the pre-made mixture as described above for anadditional 6 hours. The reaction mixture was concentrated and theresultant oil dissolved in 1.0 ml water and adjusted to pH 8 withsaturated NaHCO₃ solution. The solution was concentrated and purified bychromatography on a C-18 column (55% acetone/water) to give 300 mg (78%)of (29) as a colorless oil. ¹H NMR (CD₃OD) δ 7.40-7.25 (m, 10H), 5.11(s, 2H), 5.06 (s, 2H), 3.82 (m, 1H), 3.55 (m, 1H), 3.40-3.10 (m, 6H),1.95-1.30 (m, 8H). HRMS m/z calcd. for C₂₆H₃₆N₃O₇ 502.2553, found502.2531. [α]²⁴ _(D)+4.2° (c=1.00, CH₃OH).

EXAMPLE 17(2S,9R)-2-Amino-11-[(Benzyloxycarbonyl)amino]-7-(carbobenzyloxy)-9-(tetrahydropyran-2-yloxy)-7-azaundecanoicAcid (30)

Trifluoroacetic acid (115 mg, 1.01 mmol) was added to a solution of (29)(265 mg, 0.53 mmol) in CHCl₃ (5 ml). The solution was concentrated invacuo. The resultant oil was dissolved in dry CH₂Cl₂ (15 ml) and3,4-dihydro-2H-pyran (51 mg, 55 μl, 0.61 mmol) was added at roomtemperature. The reaction progress was monitored by TLC and threeadditional portions of 3,4-dihydro-2H-pyran (51 mg each) were added overthe next 7 hours. The reaction mixture was stirred for an additional 12hours and concentrated in vacuo. The oil was dissolved in water andmethanol (1:1, 4 ml) and adjusted to pH, 7 with saturated NaHCO₃solution. The solution was concentrated and the crude oil purified bychromatography on a C-18 column (55% acetone/water) to give 210 mg (68%)of (30) as a colorless oil and 20 mg (8%) recovered starting material(29). ¹H NMR (CD₃OD) δ 7.40-7.22 (m, 10H), 5.10 (m, 2H), 5.04 (s, 2H),1H), 4.02-3.68 (m, 2H), 3.50 (m, 1H), 3.44-3.06 (m, 7H), 1.98-1.28 (m,14H); ¹³C NMR (CD₃OD) δ 174.3, 158.7, 158.1, 138.5, 129.7, 129.6, 129.5,129.3, 129.0, 128.8, 101.7, 100.1, 74.9, 68.3, 67.4, 65.3, 56.2, 48.4,38.2, 34.3, 32.6, 32.1, 28.5, 26.4, 23.6, 21.8, 21.2. HRMS m/z calcd.for C₃₁H₄₃N₂O₈ 586.3128, found 586.3118. [α]²⁴ _(D)+4.0° (c=0.25,CH₃OH).

EXAMPLE 18 Sodium(2S,9R)-11-[(Benzyloxycarbonyl)amino]-7-(carbobenzyloxy)-2-[(9-fluorenylmethoxy-carbonyl)amino]-9-(tetrahydropyran-2-yloxy)-7-azaundecanoicAcid (31)

A solution of 9-fluorenylmethyl N-succinimidyl carbonate (181 mg, 0.53mmol) in DMF (2.5 ml) was added to a solution of (30) (210 mg, 0.36mmol) in 9% Na₂CO₃ (0.836 ml, 0.72 mmol) at 0° C. and stirred overnightat room temperature. The pH was adjusted to 7 with 0.1 N HCl. Themixture was concentrated to an oil and purified by flash chromatography(90% CHCl₃/MeOH) to give (31) (239 mg, 83%) as a colorless oil. 1H NMR(CDCl₃) 67.78 (m, 2H), 7.60 (m, 2H), 7.30 (m, 14H), 5.72 (m, 2H), 5.18(s, 2H), 5.16 (s, 2H), 4.60 (m, 1H), 4.52-4.22 (m, 3H), 4.20 (m, 11H),4.00-3.72 (m, 3H), 3.50-3.10 (m, 6H), 2.00-1.22 (m, 14H); ¹³C NMR (150MHz) (CDCl₃) δ 174.6, 156.7, 156.4, 143.9, 143.8, 141.3, 136.8, 136.5,128.5, 128.5, 128.1, 128.0, 127.9, 127.7, 127.1, 125.1, 124.9, 120.0,100.7, 73.5, 67.4, 67.2, 66.6, 53.5, 48.4, 47.2, 32.9, 32.0, 31.5, 30.8,28.0, 27.3, 25.2, 22.0, 21.1, 19.8. HRMS m/z calcd. for C₄₆H₅₃N₃O₁₀Na

A solution of 9-fluorenylmethyl N-succinimidyl carbonate (181 mg, 0.53mmol) in DMF (2.5 ml) was added to a solution of (30) (210 mg, 0.36mmol) in 9% NaCO₃ (0.836 ml, 0.72 mmol) at 0° C. and stirred overnightat room temperature. The pH was adjusted to 7 with 0.1 N HCl. Themixture was concentrated to an oil and purified by flash chromatography(90% CHCl₃/MeOH) to give (31) (239 mg, 83%) as a colorless oil. 1H NMR(CDCl₃) 67.78 (m, 2H), 7.60 (m, 2H), 7.30 (m, 14H), 5.72 (m, 2H), 5.18(s, 2H), 5.16 (s, 2H), 4.60 (m, 1H), 4.52-4.22 (m, 3H), 4.20 (m, 1H),4.00-3.72 (m, 3H), 3.50-3.10 (m, 6H), 2.00-1.22 (m, 14H); ¹³C NMR (150)MHz) (CDCl₃) δ 174.6, 156.7, 156.4, 143.9, 143.8, 141.3, 136.8, 136.5,128.5, 128.5, 128.1, 128.0, 127.9, 127.7, 127.1, 125.1, 124.9, 120.0,100.7, 73.5, 67.4, 67.2, 66.6, 53.5, 48.4, 47.2, 32.9, 32.0, 31.5, 30.8,28.0, 27.3, 25.2, 22.0, 21.1, 19.8. HRMS m/z calcd. for C₄₆H₅₃N₃O₁₀Na830.3629, found 830.3661. Anal. calcd. for C₄₆H₅₃N₃O₁₀: C68.38, H, 6.61,N, 5.20. Found: C, 68.55, H, 6.63, N, 5.26. [α]²⁶ _(D)+3.4° (c=1.00,CHCl₃).

6 1 8 PRT Homo sapiens SITE 3 Xaa = Hypusine 1 Thr Gly Xaa His Gly HisAla Lys 1 5 2 12 PRT Artificial Sequence Example of peptides ofinvention 2 Ser Thr Ser Lys Thr Gly Xaa His Gly His Ala Lys 1 5 10 3 6PRT Artificial Sequence Example of peptides of invention 3 Cys Thr GlyXaa His Gly 1 5 4 5 PRT Artificial Sequence Example of peptides ofinvention 4 Thr Gly Xaa His Gly 1 5 5 6 PRT Artificial Sequence Exampleof peptides of invention 5 Lys Thr Gly Xaa His Gly 1 5 6 6 PRTArtificial Sequence Example of a peptide of the invention 6 Cys Thr GlyXaa His Xaa 1 5

What is claimed is:
 1. In a method of preparing peptides, theimprovement comprising synthesizing a peptide containing a hypusinemoiety using a hypusine reagent having the formula:

wherein: Q₁ and Q₂ may be the same or different and are amino protectivegroups; Q₃ is H or an amino protective group which is orthogonal to theamino protective groups Q₁ and Q₂; and Z is a hydroxy protective group.2. A method according to claim 1 wherein said method of preparingpeptides comprises synthesis of a peptide chain on an insoluble support.3. The method of claim 1 wherein the absolute configuration of thecarbon atoms at the 2-position and the 9-position, respectively, of thehypusine moiety is selected from the group consisting of (2R,9S),(2S,9S) and (2R,9R).
 4. The method of claim 1 wherein the absoluteconfiguration of the carbon atoms at the 2-position and the 9-position,respectively, of the hypusine moiety is (2S,9R).
 5. The method of claim1 wherein the peptide that is synthesized comprises the hexapeptide:L-Cys-L-Thr-Gly-Hpu-L-His-Gly (SEQ ID NO: 3).
 6. The method of claim 5wherein the absolute configuration of the carbon atoms at the 2-positionand the 9-position, respectively, of the -Hpu-linkage in the peptidethat is synthesized is selected from the group consisting of (2R,9S),(2S,9S) and (2R,9R).
 7. The method of claim 5 wherein the absoluteconfiguration of the carbon atoms at the 2-position and the 9-position,respectively, of the -Hpu-linkage in the peptide that is synthesized is(2S,9R).
 8. The method of claim 1 wherein the peptide that issynthesized comprises the pentapeptide: L-Thr-Gly-Hpu-L-His-Gly (SEQ IDNO: 4).
 9. The method of claim 8 wherein the absolute configuration ofthe carbon atoms at the 2-position and the 9-position, respectively, ofthe -Hpu-linkage in the peptide that is synthesized is (2S,9R).
 10. Themethod of claim 8 wherein absolute configuration of the carbon atoms atthe 2-position and the 9-position, respectively, of the -Hpu-linkage inthe peptide that is synthesized is selected from the group consisting of(2R,9S), (2S,9S) and (2R,9R).
 11. The method of claim 1 where in thepeptide that is synthesized is L-Cys-L-Thr-Gly-Hpu-L-His-Gly-OH (SEQ IDNO: 6).
 12. The method according to claim 1 wherein the peptide chain issynthesized in solution.
 13. A method of preparing a peptide containinga hypusine moiety comprising the steps of: (a) providing a solid phasesupport; (b) providing a group of amino acid compounds comprising aminoacids and amino acid derivatives, further including peptide fragments,selectively-protected peptide fragments, selectively protected aminoacids, and a selectively- protected hypusine reagent, said hypusinereagent having the formula;

 wherein: Q₁ and Q₂ may be the same or different and are aminoprotective groups; Q₃ is H or an amino protective group which isorthogonal to the amino protective groups Q₁ and Q₂; and Z is a hydroxyprotective group; (c) contacting the solid phase support with a firstcompound selected from the group consisting of amino acids, amino acidderivatives, peptide fragments, selectively-protected peptide fragments,selectively-protected amino acids, and the selectively-protectedhypusine reagent, thereby attaching the first compound to the solidphase support; (d) sequentially contacting the first compound withadditional compounds selected from the group consisting of amino acids,amino acid derivatives, peptide fragments, selectively-protected peptidefragments, selectively-protected amino acids, and theselectively-protected hypusine reagent, including at least one compoundwhich is the selectively-protected hypusine reagent, thereby producing apeptide chain containing a hypusine moiety on the solid phase support;(e) removing the peptide chain from the solid phase support; and (f)isolating the peptide chain containing the hypusine moiety.
 14. Themethod of claim 13 wherein the first compound being added to the solidphase support has at least one functional group which is protected by aprotecting group, and wherein said protecting group is removed after thepeptide chain is completed.
 15. The method of claim 13 wherein at leastone compound being attached to the peptide chain has at least onefunctional group which is protected by a protecting group, and whereinthe protecting group is removed after the peptide chain is completed.